Gap junctions are specialized regions of the cell membrane with clusters of hundreds to thousands of densely packed gap junction channels that directly connect the cytoplasmic compartment of two neighboring cells. The gap junction channels are composed of two hemichannels (connexons) provided by each of two neighboring cells. Each connexon consists of six proteins called connexins (Cx). The connexins are a large family of proteins all sharing the basic structure of four transmembrane domains, two extracellular loops, and a cytoplasmic loop. There is a high degree of conservation of the extracellular loops and transmembrane domains among species and connexin isoforms. The length of the C-terminus, however, varies considerably giving rise to the classification of the connexins on the basis of the molecular weight. The gap junction channel can switch between an open and a closed state by a twisting motion. In the open state ions and small molecules can pass through the pore. The conduction of the electrical impulse and intercellular diffusion of signaling molecules take place through the gap junctions and normally functioning gap junctions are therefore a prerequisite for normal intercellular communication. Normal intercellular communication is essential for cellular homeostasis, proliferation and differentiation.
The link between abnormalities in connexins and disease has been established in humans as will appear in the sections below. One example is Chagas' disease caused by the protozoan parasite Trypanosoma cruzi. This disease is a major cause of cardiac dysfunction in Latin America. An altered Cx43 distribution has been observed in cells infected by Trypanosoma cruzi and this alteration may be involved in the genesis of the conduction disturbances characterizing the disease[7].
In a multicellular organism, co-ordination between cells is of paramount importance. Among the various means of cellular cross talk, gap junctions provide the most direct pathway. Gap junctions are one type of junctional complex formed between adjacent cells and consist of aggregated channels that directly link the interiors (cytoplasm) of neighbouring cells. In the adult mammal, gap junctions are found in most cell types with one known exception being circulating blood elements.
Relatively little is known about the connexin gene structure. Results reported for mouse Cx43 revealed that Cx43 contains two exons and an intron located in the 5′ untranslated region. Several putative transcription factor binding sites have been identified in the 5′ proximal promotor. In vitro studies have shown that permeable channels could be produced by hemichannels composed of different pairs of connexins. For example, Cx43 can produce functional channels with Cx32, Cx37, Cx40 and Cx45 and endogenous Cx of oocytes (Cx38) but not with Cx26 oocytes. However, very little is known about their properties as well as about the regulation of permeability of these heterochannels. Cx are expressed in the vast majority of tissues and single cells are able to express several different Cx. Permeable gap junctions can be formed between cells, which express different types of Cx. Thus the gap junction intercellular communication (GJIC) in tissues appears to be very important for maintenance of tissue integrity. It appears that several genes are making the equivalent products in order to prevent the loss of GJIC due to a mutation in one of the genes.
The pore diameter of the gap junction channel formed has been reported to be in the range of 0.8-1.4 nm. Gap junctions are relatively non-selective and allow the passage of molecules up to about 1000 Daltons. Such substances are, i.a., ions, water, sugars, nucleotides, amino acids, fatty acids, small peptides, drugs, and carcinogens. Channel passage does not require ATP and appears to result from passive diffusion. This flux of materials between cells via gap junction channels is known as gap junctional intercellular communication (GJIC), which plays an important role in the regulation of cell metabolism, proliferation, and cell-to-cell signaling. One of the most significant physiological implications for GJIC is that gap junction coupled cells within a tissue are not individual, discrete entities, but are highly integrated with their neighbors. This property facilitates homeostasis and also permits the rapid, direct transfer of second messengers between cells to co-ordinate cellular responses within the tissue.
The process of GJIC is regulated by a variety of mechanisms that can be broadly divided into major categories. In one type of regulation the cellular quantity of gap junctions is controlled by influencing the expression, degradation, cellular trafficking of connexins to the plasma membrane, or assembly of connexins into functional gap junctions. Impaired GJIC caused by the down-regulation of connexin expression, e.g. in tumor cells, is an example of this mode of regulation. Another type of regulation does not generally involve any gross alteration of the cellular levels of gap junctions or connexins, but induces opening or closure (gating) of existing gap junctions. Extracellular soluble factors, such as mitogens (e.g. DDT), hormones (e.g. catecholamines), anaesthetics (e.g. halothane), intracellular biomolecules (e.g. cAMP), and cell stress (e.g. mechanical or metabolic stress) can result in this type of regulation. Additionally, GJIC is regulated during the cell cycle and during cellular migration.
The mode of GJIC regulation or junctional gating has been widely studied for gap junctions especially gap junctions composed of Cx43. Some factors exert their inhibitory effects on GJIC indirectly, for example, by altering the lipid environment and cell membrane fluidity, whereas other GJIC inhibitors include oncogenes, growth factors, and tumor promoters, which induce various modifications of the Cx43. Disruption of junctional permeability may be necessary for mediating the specific biological functions of the latter group. These agents initiate complex signaling pathways consisting of the activation of kinases, phosphatases, and interacting proteins. understanding the mechanisms of action of these GJIC modulators will not only define their respective signaling pathways responsible for junctional regulation, but will also provide experimental tools for characterising the biological functions of GJIC and connexins. Changes in the phosphorylation of specific sites of the cytoplasmic carboxy terminal domain of Cx43 appear to be pivotal to the opening and closing of the gap junctional channel. Phosphorylation of the carboxy terminal domain may also be important to the process of bringing Cx43 gap junctional hemicomplex to the cell membrane, its internalisation and degradation. Connexins have half-lives (hours) that are much shorter than most plasma membrane proteins (days), e.g. the half-life of Cx43 in rat heart is less than 1½ hour. Thus, regulation of the turnover rate would be an important factor in regulating GJIC.
The carboxy terminal domain contains putative phosphorylation sites for multiple protein kinases (PKA, PKC, PKG, MAPK, CaMkII and tyrosine kinase). Phosphorylation of these sites of the carboxy terminal domain results in closure of gap junctional channels and various inhibitors of Cx43 gap junctional channels use different signalling pathways to induce phosphorylation of the carboxy terminal domain. The cell type and the particular inhibitor determine which signalling pathways to be used and the type of the involved protein kinase points to the intracellular messenger system utilised. Thus activation of PKA requires involvement of the cAMP second messenger system while PKC requires involvement of the phosphoinositol intracellular signalling system.
Other mechanisms regulating channel gating include intracellular levels of hydrogen and calcium ions, transjunctional voltage, and free radicals. Decreased pH or pCa induce channel closure in a cell- and connexin-specific manner.
Many physiological roles besides growth control have been proposed for GJIC. Homeostasis: GJIC permits the rapid equilibration of nutrients, ions, and fluids between cells. This might be the most ancient, widespread, and important function for these channels. Electrical coupling: Gap junctions serve as electrical synapses in electrically excitable cells such as cardiac myocytes, smooth muscle cells, and neurones. In these tissues, electrical coupling permits more rapid cell-to-cell transmission of action potentials than chemical synapses. In cardiomyocytes and smooth muscle cells, this enables their synchronous contraction. Tissue response to hormones: GJIC may enhance the responsiveness of tissues to external stimuli. Second messengers such as cyclic nucleotides, calcium, and inositol phosphates are small enough to pass from hormonally activated cells to quiescent cells through junctional channels and activate the latter. Such an effect may increase the tissue response to an agonist. Regulation of embryonic development: Gap junctions may serve as intercellular pathways for chemical and/or electrical developmental signals in embryos and for defining the boundaries of developmental compartments. GJIC occurs in specific patterns in embryonic cells and the impairment of GJIC has been related to developmental anomalies and the teratogenic effects of many chemicals.
The intercellular communication ensures that the activities of the individual cells happen in a co-ordinated fashion and integrates these activities into the dynamics of a working tissue serving the organism in which it is set. It is therefore not very surprising that a wide variety of pathological conditions have been associated with decreased GJIC. The link between abnormalities in connexins and a range of disease states has been established both in vitro and in vivo. One example is regulation of gap junctional communication by a pro-inflammatory cytokine in airway epithelium, where Chanson M, Berclaz P Y, Scerri I, Dudez T, Wernke-Dollries K, Pizurki L, Pavirani A, Fiedler M A, Suter S. (Am J Pathol 2001 May;158(5):1775-84) found that decreased intercellular communication induced by TNF-alpha progressively led to inflammation.
In summary, there is plenty of evidence linking malfunction, such as gating or closure or even absence of gap junctions to an increased risk of disease. No currently available drug for the treatment of said diseases acts as a facilitator of intercellular communication by facilitating or increasing gap junction function. However a group of peptides (the antiarrhythmic peptides) capable of increasing gap junction conductance has been described in the past. A summary is presented in PCT/DK01/00127 which is hereby incorporated by reference. A summary of the present invention is disclosed in U.S. Ser. No. 09/792,286 as filed on Feb. 22, 2001. The disclosure of the U.S. Ser. No. 09/792,286 is incorporated herein by reference.
The antiarrhythmic peptides are a group of peptides that exert their effect selectively on gap junctions and thus decrease cellular uncoupling and also reduce dispersion of action potential duration. However, the native AAP as well as the synthetic AAP10 possess several undesired features, such as, low stability, high effective concentration etc. that has hitherto prevented their utilisation as drugs. Grover and Dhein[21] have characterised two semi cyclic conformations of AAP10 using nuclear magnetic resonance spectroscopy. Therefore, one approach to obtaining a stable antiarrhythmic peptide could be the provision of cyclic derivatives of antiarrhythmic peptides. DE19707854 discloses apparently cyclic CF3C(OH)-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH (SEQ ID NO: 1) and cyclic CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH (SEQ ID NO: 1) having the same antiarrhythmic properties as AAP and AAP10, but stated to have improved stability in aqueous solution and after repeated cycles of freezing and thawing. However, the experimental conditions described in DE19707854 are insufficient for the preparation of said cyclic compounds, and the chemical identification data given therein using HPLC is not sufficient for identification of said cyclic compounds. U.S. Pat. No. 4,775,743 discloses HP5, a peptide derivative having the sequence N-3-(4-hydroxyphenyl)propionyl-Pro-4Hyp-Gly-Ala-Gly-OH (SEQ ID NO: 2) and being active against platelet agglutination. Dhein and Tudyka[22] have reviewed the literature on peptides including peptide derivatives belonging to the group of antiarrhythmic peptides for activity and concentration, cf. table 1 therein, and found only 7 compounds to be active and further 4 compounds to be weakly active. However, none of these peptides or peptide derivatives have been shown to be sufficiently stable to be effective in a therapy regimen.
The peptides herein increase gap junction intercellular communication (GJIC) in vertebrate tissue, and specifically in mammalian tissue, and are useful in the treatment of a wide spectrum of diseases and ailments in vertebrates, such as mammals, relating to or caused by a decreased function of intercellular gap junction communication as is described below.
Thus, it is a purpose of the present invention to provide a method of preventing or treating diseases and medical conditions that are characterised in reduced or impaired cellular communication, such as caused by impaired gap junctional intercellular communication or impaired coupling through gap junctions. Examples of diseases and medical conditions are inflammation of airway epithelium, disorders of alveolar tissue, bladder incontinence, impaired hearing due to diseases of the cochlea, endothelial lesions, diabetic retinopathy and diabetic neuropathy, ischemia of the central nervous system and spinal cord, dental tissue disorders including periodontal disease, kidney diseases, and failures of bone marrow transplantation as mentioned above.